Our published data and the preliminary data presented in this application have demonstrated that hyperglycemia suppresses AMPK, which in turn perpetuates oxidative stress and vascular injury in diabetes. The central hypothesis of this application is that AMPK12 inhibition increases 26S activity, which increases I:B1 degradation, p65 nucleus localization, and consequent NF:B activation. Activated NF:B binds to the promoter of NAD(P)H oxidase subunits, including NOX4, gp91phox(NOX2), p67phox, p47phox, resulting in an increase in NAD(P)H oxidase activity and ROS, which causes endothelial cell dysfunction and accelerated atherosclerosis. Chronic proteasome inhibition of the 26S proteasome prevents endothelial dysfunction and atherogenesis by inhibiting proteasome-dependent NF:B-mediated activation of NAD(P)H oxidase. This hypothesis will be tested in three interrelated aims. These first two aims are designed to establish whether chronic hyperglycemia inhibition AMPK functions (Aim #1) and whether AMPK inhibition causes abnormal expression of adhesion molecules and oxidant stress in diabetes (Aim #2). Finally, we will test the contributions of RNS-mediated AMPK inhibition in the development of accelerated atherosclerosis in diabetes (Aim #3). Although the in vivo relationships among atherosclerosis, oxidant stress, and AMPK in diabetes are undoubtedly complex, the emerging role for AMPK in oxidant stress and atherosclerosis warrant further study. The studies proposed here represent the first set of definitive studies to determine the role of ONOO- and AMPK inhibition in the pathogenesis of diabetic vascular dysfunction and atherosclerosis. As such, they have the potential to explain the long recognized decrease in AMPK that occurs in animals and human patients with diabetes. We believe that the proposed studies will provide novel information as to how the metabolic stress associated with diabetes causes damage to the endothelium and how the endothelial cell attempts to protect itself against these stresses and whether ONOO- or AMPK are potential targets for therapy.